In vivo thromboembolic stroke model performance
Adult male Swiss mice (Jackson labs, Bar Harbor, Me) with a mean weight of 30 g were used for this study. All procedures were performed in accordance with the European Communities Council Directive (86/609/EEC) and approved by the Ethics Committee on Animal Welfare of University Complutense (PROEX No. 016/18) and are reported according to ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Animals were housed individually under standard conditions of temperature and humidity and at a 12 h light/dark cycle (lights on at 8 h) with free access to food and water.
The surgical procedure for in situ thromboembolic model was carried out during light cycle (9h-13h) as previously described [20, 21]. Briefly, mice were anaesthetized in a chamber ventilated with 2.5% isoflurane and then maintained at 1.5–2% isoflurane in a 30/70% mixture of O2/air. Body temperature was maintained at 36.5–37°C using a feedback-controlled heating blanket. Mouse alpha-thrombin (2 UI) was injected into the MCA to induce its occlusion by a clot. A clot was defined as stable when laser Doppler flowmetry displayed a drastic fall of brain perfusion (mean reduction of 70– 80%) that remained stable during 60 min. For reperfusion, rt-PA (10 mg/kg) was intravenously administered 3 h after thrombin injection. We considered that reperfusion was effective when blood flow was recovered (in the range of 60–100% of basal values) and remained stable within the first 60 min after rt-PA injection.
A total of 32 animals were assigned arbitrarily to three groups: (1) middle cerebral artery occlusion (MCAO) (n = 12), in which the middle cerebral artery (MCA) was permanently occluded and vehicle was intravenously administered 3 h after thrombin injection, (2) MCAO + rt-PA (n = 11), in which artery recanalization was achieved administering rt-PA at 3 h after thrombin injection, and (3) sham (n = 9), in which the MCA was surgically exposed but not occluded. In vivo data analysis was performed by a person other than the experimenter and sample size estimation was based in previous studies. Mice with spontaneous reperfusion (without rt-PA administration) (n = 3), with extraparenchymal hemorrhages (n = 2) or with striatal lesions (n = 1) were excluded of further analysis. No spontaneous mortality was found after MCAO and this was unaffected by the rt-PA administration.
Blood sample collection
Blood tail samples were collected before (t = pre-MCAO) and after the experimental procedure (3 h post-thrombin injection, previously to rt-PA or vehicle administration (t = 0), and at 3 and 24 h after rt-PA or vehicle injection) in the different experimental groups (Fig. 1a). Samples were kept at room temperature for 1 h and at 4°C overnight, allowing coagulation. Samples were then centrifuged at 1500g and 4°C and the obtained serum was kept at 80°C until its analysis.
Measurement of serum Cav-1 levels
Serum Cav-1 concentration was measured at pre-MCAO, 0, 3 and 24 h using an ELISA kit (SEA214Mu, Cloud Clone Corp. Houston, USA) in accordance with the manufacturer’s instructions. Data were divided by the corresponding pre-MCAO level and represented as a percentage to reduce initial variability. The time point of 0 h was taken as the baseline levels.
Tissue collection
Twenty-four hours after MCAO (Fig. 1a), mice were sacrificed by an overdose of sodium pentobarbital and were transcardially perfused with 0.1 M phosphate buffer (pH 7.4) followed by a solution of 4% paraformaldehyde in PBS. Brains were post-fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) solution overnight and then placed in 30% sucrose in PBS for 3 days at 4ºC until they sank. Brains were then frozen in isopentane and stored at -80ºC for further analysis.
Determination of brain edema, infarct volume and volume of hemorrhage
One coronal section of 30 µm thickness every 400 µm was stained with cresyl violet (Nissl immunostaining) and diaminobenzidine (DAB) to measure edema and infarct and hemorrhagic volumes as previously reported [20]. Briefly, the ratio of the entire area of the ipsilateral hemisphere to that of the contralateral one was considered as edema. The infarct area was delineated and determined (in mm2) by counting the number of pixels within the outline. The infarct volume (in mm3) was calculated as the sum of the orthogonal projections of each damaged area over the section thickness. In order to exclude the brain swelling effects, infarct volume was corrected by the edema and data were expressed as a percentage of the hemisphere. All noticeable hemorrhages, both petechial and parenchymal ones, were quantified by stereology using Cast Grid software (Visiopharm, Denmark). The volume of extravasated red cells was calculated by Cavalieri applying the following formula: (volume = a(p)·d· P) where a(p) is the area associated to the dot, d the distance between two consecutive sections, and P the counted dots inside the hemorrhage.
Immunohistochemistry
Serial coronal sections of cryopreserved brain (15 µm-thick) were obtained in a cryostat (CM1950, Leica) at 23°C, at coordinates between Bregma 2.4 mm and − 4.2 mm. The slices were mounted onto SuperFrost/Plus slides (Menzek-Gläser, Braunschweig, Germany) and stored at -80°C until immunohistochemistry staining.
Four double labelling of Cav-1 and extravasated IgG, frozen sections were dried and permeabilized with TBS 0.5% Triton X-100 for 10 min and blocked with TBS-T (TBS 0.1% Triton X-100) 1% BSA for 30 min. Sections were then incubated with a rabbit anti-caveolin-1 antibody (1:200, sc-894, Santa Cruz Biotechnology) for 3 h at room temperature, washed 3 times with TBS-T and incubated 2 h more with Alexa Fluor® 488 goat anti-rabbit and 594 goat anti-mouse IgGs (1:750 and 1:100 respectively, Invitrogen). Finally, samples were washed, stained with DAPI and mounted with Dako fluorescent mounting medium (Dako North America Inc., USA). No immunostaining was observed in control slides without the primary or secondary antibodies. Mouse IgG staining was used to identify the infarcted zone where the BBB leakage occurs [22].
Additionally, a similar immunohistochemistry protocol was used to confirm the expression of Cav-1 in brain endothelial cells. Sections were simultaneously incubated with the rabbit anti-caveolin-1 antibody and the rat anti-PECAM-1 antibody (1:50, sc-18916, Santa Cruz Biotechnology) for overnight at 4ºC and the Alexa Fluor® 488 goat anti-rabbit and 594 goat anti-rat (1:500, A-11007, Invitrogen) for 2 h at room temperature, respectively.
Microphotographs were taken with an Olympus DP70 digital camera (Japan) attached to a BX41 Olympus microscope. Image-J image analysis software was used to assess greyscale intensity levels. An average of Cav-1 intensity was measured in the ipsilateral hemisphere (infarcted zone) and in the contralateral hemisphere (CTR) between Bregma 2.4 mm and − 4.2 mm for all regions. In the sham group, anatomically equivalent brain areas in the ipsilateral and contralateral hemispheres were analyzed.
Cell culture
Immortalized mouse brain endothelial cell line (bEnd.3) purchased from ATCC (CRL-2299), were seeded in 60mm Petri dishes (Corning, USA) for western blot analysis, or on the top of a transwell insert (0.3 cm2 surface area, 0.4 µm pore size, PET membrane, BD Falcon), for metabolic activity and transcelullar permeability analysis, as previously described [23]. bEnd.3 cells were grown as a monolayer in DMEM high glucose (HG) medium with 1% glutamine (Gibco, USA), 10% fetal bovine serum (Gibco, USA) and 1% Penicillin/Streptomycin (HyClone Laboratories, USA). All bEnd.3 cells used for these experiments were cultured between 25 and 30 passages, which have been shown to maintain excellent BBB characteristics in vitro [24].
OGD exposure
To mimic acute ischemia-like conditions in vitro, bEnd.3 cells were exposed to OGD for 2.5 h as we described previously [23, 25]. In brief, after overnight starvation in DMEM HG with 1% fetal bovine, bEnd.3 monolayers were subjected to OGD. The medium was replaced with glucose-free DMEM without FBS (Gibco, USA) previously perfused with N2 to purge the oxygen. The cells were then placed into a 37°C humidified hypoxic chamber with a constant N2 flow of 1 L/min and 0.15 bar pressure for 2.5 h. Regarding the control (CTR) group, the same procedure was carried out with the difference that the glucose-free medium was supplemented with glucose (5.5 mM) and incubated at 37°C with 5% of CO2. At the end of the OGD period, the media were removed and replaced with DMEM HG medium containing 10% FBS and with or without rt-PA at a concentration of 20 µg/ml and cultures were returned to the normoxic incubator. As reported in previous publications, we used 20 µg/ml of rt-PA, based on the finding that such a concentration can be observed in blood [26].
Metabolic activity and transcellular permeability analysis
bEnd.3 metabolic activity and transcellular permeability were assessed after 72 h of reoxygenation, with and without rt-PA treatment, using 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay and the passage of fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) across the cell monolayer, respectively, as previously described [23].
MTT assay was performed as follows. The medium of the transwells was aspirated, 100 µl of fresh medium and 10 µl of MTT (5mg/ml) (Sigma) were added to each transwell and cells were incubated at 37°C for 2 h. The medium was then carefully removed, and formazan crystals were lysed in 100 µl of DMSO by gently shaking the plate. Absorbance was measured at 570 nm using the SpectraMax 340PC384 Microplate Reader (Molecular Devices). MTT results were expressed as a percentage of the value in the control group.
For the analysis of transcellular permeability inserts were transferred into new wells containing 0.75 ml of fresh serum-free medium and the medium of the luminal chamber was replaced with 0.15 ml of medium containing 0.35 mg/mL of FITC-BSA. After 1 h, the abluminal medium was sampled (duplicates of 200 µl) and fluorescence was measured on a Cytation™ 5 Cell Imaging Multi-Mode Reader (Biotek) at excitation and emission wavelengths of 485 and 520 nm. Changes in permeability were calculated relative to inserts without cells (blank inserts), which served as a reference for maximum permeability. The following formula was used: permeability (% of max) = ((FITC reading of experimental insert-average FITC reading of the vehicle group)/(FITC reading of the blank insert-average FITC reading of the vehicle group)) x100.
Western blot analysis
At 0, 3, 24 and 72 h post-reoxygenation cells were collected and protein was isolated using lysis buffer (Cell Signaling, The Netherlands) (Fig. 1b). The protein concentration was measured using the BCA method (Thermo Fisher Scientific, USA). Protein samples (10 µg) were loaded and separated by electrophoresis on 4–15% Criterion™ TGX Stain-Free™ Precast Gels (Bio-Rad) at 120 V for 80–90 min. Proteins were then transferred to PVDF membranes at 30 V overnight at 4°C. After 1 h of blocking with Tris buffered saline with 0.1% Tween-20 (TBST) 5% BSA (EMD Millipore, USA), membranes were incubated with anti-phospho-caveolin-1 (Tyr14) (1:1000, Cell Signaling, The Netherlands), anti-caveolin-1 (sc-894) (1:40000, Santa Cruz Biotechnology, USA) and anti-rabbit HRP-conjugated (1:10000, Cell Signaling, The Netherlands) antibodies in TBST 3% BSA. Stripping was performed to reprove the membranes. Protein bands were revealed using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, USA). Quantification of the results was performed using Alpha Ease FC software (Alpha Innotech, USA) to measure integrated density of bands after background subtraction. Normalized expression of pCav-1 were obtained by comparing to total expression of Cav-1. Total expression of protein in the same lane were used as a loading control. These expression were obtained after exposition to 5 minutes with UV light in order to activate the trihalo compounds presents in the used criterion stain-free gels according to the previously described [25].
Cav-1 immunofluorescence in bEnd.3 cells
After 72h of reoxygenation, bEnd.3 cells were fixed with PBS 3.7% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, US) and permeabilized with PBS 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO, US) (Fig. 1b). Cells were then blocked with PBS 3% BSA and incubated 1 h with primary antibody anti-caveolin-1 (sc-894) (1:100, Santa Cruz Biotechnology) and 1 h with secondary antibody Alexa Fluor® 488 goat anti-rabbit IgG (1:40, Invitrogen) diluted in PBS 3% BSA. Finally, the nuclei were stained with DAPI. Images were captured with different channels for Alexa Fluor-488 and DAPI on a BD Pathway 855 Bioimager System (Becton–Dickinson Biosciences). Merging images were obtained in accordance with the recommended assay procedure using BD Attovision software. Total intensity of Cav-1 was quantified using Image-J 1.43 (http://rsb.info.nih.gov/ij/) software (NIH, Bethesda, MD).
Statistics
SPSS software (IBM SPSS Statistics 22) was used to perform the statistical analysis. Shapiro-Wilk test was performed to assess the normality of the data. Cav-1 immunoreactivity, metabolic activity, transcellular permeability and Cav-1 and pCav-1 in vitro expression were compared by one-way ANOVA followed by a Bonferroni post-hoc analysis when required. Analyses of Cav-1 serum levels were conducted with a linear mixed model, which corresponded to two between-group factors, the GROUP (sham, MCAO and MCAO + rt-PA) and the TIME (0, 3 and 24 h post-treatment). Bonferroni post-hoc contrast was used when required. Correlations between variables were estimated using the Spearman test. The significance level (alpha) for all tests was set at .05.